Optogenetic chloride loading in neurons : implications for epilepsy

Abstract

Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory, if intraneuronal chloride [Cl-]i rises. It has proved difficult, however, to separate out the role of raised [Cl-]i from other contributory factors in complex network phenomena such as epileptic pathology. To address this, in this thesis I used Halorhodopsin to load clusters of pyramidal cells artificially with Cl-, to explore the implication of raised [Cl-]i in the generation of epileptic activity and seizure initiation. Brief Halorhodopsin activation caused substantial positive shifts in the GABAergic reversal potential (EGABA). At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high frequency oscillations with evidence of out-of-phase firing. Such firing patterns can arise from quite small shifts in the mean [Cl-]i level, within heterogeneous neuronal populations. Notably, however, chloride-loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, sub-epileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that in vitro, chloride-loading has an adjunctive role in ictogenesis. Additionally, in this thesis an optogenetic approach to extrude Cl- was also explored, as a potential way to correct the raised [Cl-]i issue in epilepsy. A strategy that was proved to be successful was the combination of ArchaerhodopsinT (ArchT) hyperpolarizing effect and the opening of light activated Cl- channel (ChloC), which together provides a driving force and a conductance for an outward Cl- flow. Co-activation of the two opsins consistently induced a negative shift in EGABA indicative of Cl- extrusion mechanism, which in the future could be tested as a new optogenetic approach to control epilepsy.